Hydration state change of proteins upon unfolding in sugar solutions

Change in hydration number of proteins upon unfolding, Δn, was obtained from the analysis of thermal unfolding behavior of proteins in various sugar solutions with water activity, aW, varied. By applying the reciprocal form of Wyman–Tanford equation, Δn was determined to be 133.9, 124.1, and 139.2 per protein molecule for ribonuclease A at pH = 5.5, 4.2, and 2.8, respectively, 201.4 for lysozyme at pH = 5.5, and 100.1 for α-chymotripnogen A at pH = 2.0. Among the sugars tested, reducing sugars gave the lower apparent Δn as compared with nonreducing sugars probably because of the direct interaction of reducing terminal with amino group of proteins at a high temperature. From the knowledge of Δn, a new thermodynamic model for protein stability was proposed with explicit consideration for hydration state change of protein upon unfolding. From this model, the contribution of aW was proven to be always positive for stabilization of proteins and its effect is not negligible depending onΔn and aW.